Effects of flanking regions on DNA i-motif folding and stability

i-Motifs (iMs) are four-stranded non-canonical nucleic acid secondary structures that are formed by cytosine-rich sequences. Putative iM-forming sequences are concentrated in human promoter and telomeric regions, suggesting possible biological roles. However, many iMs do not readily fold at neutral pH, sparking interest in factors that may stabilize them. We performed a systematic study on how the nucleotides flanking iMs affect their stabilities and folding kinetics. We found that the mere presence of flanking nucleotides led to dramatically slower folding and lower stability compared to isolated iMs. Conversely, complementary flanking nucleotides that comprise an inverted repeat and form a hairpin with the iM in the loop led to greater stability and faster folding than the iM on its own. A bioinformatic analysis of human promoter regions showed that the flanking regions are more likely than average to be complementary to each other, suggesting that this stabilization might be biologically relevant. We analyzed several naturally occurring iM sequences and found that complementary flanking regions substantially stabilized the structures (up to 64-fold faster folding and 17.2°C more stable). Our results show that the regions of DNA flanking iMs are an important and hitherto overlooked factor in iM folding and stability.